WO2018092867A1 - Solid support containing igg-binding peptide, and method for separating igg - Google Patents
Solid support containing igg-binding peptide, and method for separating igg Download PDFInfo
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- WO2018092867A1 WO2018092867A1 PCT/JP2017/041404 JP2017041404W WO2018092867A1 WO 2018092867 A1 WO2018092867 A1 WO 2018092867A1 JP 2017041404 W JP2017041404 W JP 2017041404W WO 2018092867 A1 WO2018092867 A1 WO 2018092867A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
- C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
- C07K17/10—Peptides being immobilised on, or in, an organic carrier the carrier being a carbohydrate
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
- C07K17/06—Peptides being immobilised on, or in, an organic carrier attached to the carrier via a bridging agent
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
Definitions
- the present invention relates to a solid phase carrier comprising an IgG-binding peptide, an IgG separation column comprising the solid phase carrier, a kit comprising the solid phase carrier or column, an IgG purification method using the solid phase carrier or column, and the like.
- IgG antibody is one of the biopharmaceuticals that are currently attracting the most attention.
- antibody drugs centered on IgG antibodies have come to be used in the pharmaceutical field, and their importance in industrial and pharmaceutical use is increasing.
- Protein A columns play a central role in antibody purification, and many antibody drug manufacturers have introduced purification systems centered on these columns.
- Protein A column has some problems.
- Protein A is a bacterial protein, has high immunogenicity after human administration, and is endangered by endotoxins.
- As an affinity ligand used for the purification of pharmaceuticals such as IgG protein A as a ligand is required to have a high degree of purification so that inconvenient contamination does not occur.
- IgG can be purified by a peptide ligand containing a specific sequence cyclized by a disulfide bond (Patent Document 1). This peptide ligand is washed with an alkaline solution. There was a problem that the affinity decreased due to the repetition of.
- An object of the present invention is to provide an IgG-binding peptide that can be used for IgG purification and has excellent stability, for example, alkali stability. Moreover, this invention makes it a subject to provide the method etc. which refine
- the present inventor has found that the stability of the peptide is remarkably improved by crosslinking the sulfide group at the cysteine residue in the peptide with a linker having a specific structure, and has completed the present invention.
- a solid phase carrier immobilizing a peptide characterized by being capable of binding to human IgG Said peptide is represented by the following formula I: (X 1-3 ) -C- (X 2 ) -H- (Xaa1) -G- (Xaa2) -LVWC- (X 1-3 ) (I)
- each X is independently any amino acid residue other than cysteine, C is a cysteine residue; H is a histidine residue;
- Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
- G is a glycine residue;
- Xaa2 is a glutamic acid residue or an asparagine residue, L is a leucine residue; V is a valine residue, and W is a tryptophan residue.
- the sulfide group in the two cysteine residues outside the peptide comprising an amino acid sequence consisting of 13-17 amino acid residues represented by A solid phase carrier linked by a linker selected from the group consisting of: wherein R is a substituted or unsubstituted C1-C6 alkyl.
- each X is independently any amino acid residue other than cysteine, C is a cysteine residue; H is a histidine residue; Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof, G is a glycine residue; Xaa2 is a glutamic acid residue or an asparagine residue, L is a leucine residue; V is a valine residue; W is a tryptophan residue; Xaa3 is an alanine residue, a serine residue or a threonine residue, and Xaa4 is a tyrosine residue or a tryptophan
- the solid phase carrier according to (1) comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by: (3)
- the peptide is represented by the following formula III: (X 1-3 ) -CAYH- (Xaa1) -GELVWC- (X 1-3 ) (III)
- each X is independently any amino acid residue other than cysteine, C is a cysteine residue; A is an alanine residue, Y is a tyrosine residue; H is a histidine residue; Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof, G is a glycine residue; E is a glutamic acid residue, L is a leucine residue; V is a valine residue, and W is a tryptophan residue.
- the solid phase carrier according to (1) or (2) comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by: (4)
- the peptide is composed of any one of the following amino acid sequences 1) to 14), wherein Xaa1 is an arginine residue, a lysine residue, a leucine residue, an asparagine residue, or a derivative thereof: , Xaa2 is homocysteine, the solid phase support according to (4): 1) DCAYH (Xaa1) GELVWCT (SEQ ID NO: 1) 2) GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2) 3) RCAYH (Xaa1) GELVWCS (SEQ ID NO: 3) 4) GPRCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 4) 5) SPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 5) 6) GDDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 6) 7) GPSCAYH (Xaa1) GELVWCTFH (SEQ ID NO
- the peptide is represented by the following formula IV: DC- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -LVWCT (IV) (Where D is an aspartic acid residue; C is a cysteine residue; H is a histidine residue; Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof, G is a glycine residue; Xaa2 is a glutamic acid residue or an asparagine residue, L is a leucine residue; V is a valine residue; W is a tryptophan residue; T is a threonine residue; Xaa3 is an alanine residue or a threonine residue, and Xaa4 is a tyrosine residue or a tryptophan residue.
- the peptide is composed of any one of the following amino acid sequences 1) to 4), wherein Xaa1 is an arginine residue, a lysine residue, a leucine residue, an asparagine residue, or a derivative thereof: , (6) solid phase carrier: 1) DCTYH (Xaa1) GNLVWCT (SEQ ID NO: 15) 2) DCAYH (Xaa1) GNLVWCT (SEQ ID NO: 16) 3) DCTYH (Xaa1) GELVWCT (SEQ ID NO: 17) 4) DCAWH (Xaa1) GELVWCT (SEQ ID NO: 18).
- a solid phase carrier immobilizing a peptide that is capable of binding to human IgG Said peptide is represented by the following formula V: DC- (Xaa2)-(Xaa3)-(Xaa4)-(Xaa1) -G- (Xaa5) -L- (Xaa6) -WCT (V) (Where D is an aspartic acid residue; C is a cysteine residue; G is a glycine residue; L is a leucine residue; W is a tryptophan residue; T is a threonine residue; Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof, Xaa2 is an alanine residue, a serine residue or a threonine residue, Xaa3 is a tryptophan residue or a tyrosine residue, Xaa4 is a histidine residue,
- Xaa1 is an arginine residue, a lysine residue or an acylated derivative thereof, or a leucine residue.
- the linker is represented by the following formula: The solid phase carrier according to any one of (1) to (10), which is a linker represented by the formula: (12) The solid phase carrier according to any one of (1) to (11), wherein the N-terminus of the peptide is PEGylated.
- the stability of the peptide contained in the solid phase carrier of the present invention is improved by crosslinking the sulfide group in the cysteine residue with a linker having a specific structure. Therefore, the solid phase carrier of the present invention can be used for efficient IgG purification because the IgG binding ability is hardly reduced by an alkali washing step or the like.
- FIG. 1 shows the results when human serum-derived ⁇ -globulin (Wako) was adsorbed to the peptide-immobilized column prepared in Example 3 and eluted using an acidic elution solution (20 mM mM citric acid, pH 2.5). Indicates. The horizontal axis represents the volume of the eluate, and the vertical axis represents the protein concentration measured by absorbance at 280 nm.
- FIG. 2 shows the result of SDS-PAGE of each fraction obtained by eluting the protein with citric acid or IgG-binding peptide from the peptide-immobilized column prepared in Example 3, and fractionating 0.5 mL. Protein detection was performed by CBB staining.
- FIG. 3 shows that after equilibrating three kinds of columns (1 mg, 4 mg, 10 mg) with different amounts of peptide immobilization with an adsorption solution, 1 mg of 1 mg / mL human serum-derived ⁇ -globulin dissolved in the adsorption solution
- the DBC measurement results are shown when the liquid is fed at a flow rate of / min (residence time of 1 min).
- the DBC was determined from the amount of sample sent until the value excluding non-adsorbed components reached 10% of the absorbance of the entire sample at 280 nm absorbance.
- FIG. 4 shows the results of DBC measurement during sodium hydroxide treatment of the IgG-binding peptide crosslinked with the linker prepared in Example 2 and the IgG-binding peptide crosslinked with disulfide prepared in Comparative Example 1.
- Solid phase carrier in the present specification is not limited, but includes inorganic carriers such as glass beads and silica gel, synthetic polymers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, and crosslinked polystyrene, and crystallinity.
- examples include organic carriers composed of polysaccharides such as cellulose, crosslinked cellulose, crosslinked agarose, and crosslinked dextran, and organic-organic, organic-inorganic composite carriers obtained by a combination thereof, among which hydrophilic carriers are not.
- hydrophilic carrier refers to a carrier having a contact angle with water of 60 ° or less when the compound constituting the carrier is formed into a flat plate shape.
- Such carriers include polysaccharides such as cellulose, chitosan, dextran, polyvinyl alcohol, saponified ethylene-vinyl acetate copolymer, polyacrylamide, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylic acid grafting
- Representative examples include carriers made of polyethylene, polyacrylamide grafted polyethylene, glass and the like.
- the form of the solid phase carrier can be any of beads, fibers, particles, membranes (including hollow fibers), gels, etc., and any form can be selected.
- a bead shape is particularly preferably used because of easy preparation of a carrier having a specific exclusion limit molecular weight.
- the average particle size of the beads is 10 to 2500 ⁇ m, and is particularly preferably in the range of 25 ⁇ m to 800 ⁇ m from the viewpoint of easy IgG-binding peptide immobilization reaction.
- Specific examples of the solid phase carrier include magnetic beads, glass beads, polystyrene beads, silica gel beads, and polysaccharide beads.
- a functional group that can be used for the immobilization reaction of the IgG-binding peptide on the surface of the solid phase carrier, it is convenient for immobilization of the IgG-binding peptide.
- these functional groups include hydroxyl groups, amino groups, aldehyde groups, carboxyl groups, thiol groups, silanol groups, epoxy groups, succinimide groups, N-hydroxysuccinimide groups, acid anhydride groups, iodoacetyl groups, and the like. Can be mentioned.
- GCL2000 and GC700 which are porous cellulose gels
- Sephacryl® S-1000 in which allyldextran and methylenebisacrylamide are covalently crosslinked
- Toyopearl which is an acrylate carrier
- Sepharose CL4B which is an agarose crosslinking carrier
- epoxy examples include Eupergit C250L, which is a polymethacrylamide activated with a group, and an NHS activated prepacked column including a Sepharose carrier activated with an NHS group.
- the present invention is not limited to these carriers and activated carriers.
- the above-mentioned solid phase carriers may be used alone or in combination of any two or more. Further, the solid phase carrier preferably has a large surface area in view of its purpose and method of use, and preferably has a large number of pores of an appropriate size, that is, is porous.
- the IgG-binding peptide described herein is immobilized on the solid phase carrier, and the peptide can be immobilized by a method well known to those skilled in the art, for example, physical adsorption. It can be carried out by the method, the covalent bond method, the ion bond method and the like.
- the immobilization is preferably performed, for example, by covalently binding the N-terminal amino group of the IgG-binding peptide directly or via a spacer to the solid phase carrier.
- a hydrophilic spacer is not particularly limited.
- the method and conditions for immobilizing the IgG-binding peptide introduced into the solid phase carrier and the organic compound used as the spacer are not particularly limited, but are generally used when immobilizing proteins and peptides on the carrier. Is illustrated.
- the carrier is activated by reacting with a compound containing an amino group, a compound containing an N-hydroxysuccinimidyl group, cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, tresyl chloride, hydrazine, etc.
- the solid phase carrier containing the IgG-binding peptide described in the present specification can be packed in a chromatography column or the like and used to purify or separate human IgG.
- IgG refers to mammals such as primates such as humans and chimpanzees, laboratory animals such as rats, mice, and rabbits, and domestic animals such as pigs, cows, horses, sheep, and goats, In addition, it refers to IgG of pet animals such as dogs and cats, preferably human IgG (IgG1, IgG2, IgG3 or IgG4).
- the IgG in the present specification is more preferably human IgG1, IgG2, or IgG4, or rabbit IgG, and particularly preferably human IgG1, IgG2, or IgG4.
- the IgG binding peptide that can be included in the solid phase carrier of the present invention has the following formula I (X 1-3 ) -C- (X 2 ) -H- (Xaa1) -G- (Xaa2) -LVWC- (X 1-3 ) (I)
- each X is independently any amino acid residue other than cysteine
- C is a cysteine residue
- H is a histidine residue
- Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof
- G is a glycine residue
- Xaa2 is a glutamic acid residue or an asparagine residue
- L is a leucine residue
- V is a valine residue
- W is a tryptophan residue.
- the sulfide group in the two cysteine residues outside the peptide comprising an amino acid sequence consisting of 13-17 amino acid residues represented
- the notation of X 1-3 at the N-terminal or C-terminal means that 1 to 3 consecutive amino acid residues X other than cysteine (C or Cys) are independent, The amino acid residues constituting are the same or different residues, but preferably all three are composed of sequences that are not the same.
- X 2 also means that two arbitrary amino acid residues X other than cysteine (C or Cys) are consecutive, and the amino acid residues constituting them are the same or different residues. Preferably, however, the two consecutive amino acid residues consist of sequences that are not the same residue.
- the peptide represented by the formula I ′ is (X 1-3 ) -C- (X 1 ) -YH- (Xaa1) -GNLVWC- (X 1-3 ) (I ')
- each X is independently any amino acid residue other than cysteine
- C is a cysteine residue
- Y is a tyrosine residue
- H is a histidine residue
- Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof
- G is a glycine residue
- N is an asparagine residue
- L is a leucine residue
- V is a valine residue
- W is a tryptophan residue.
- an amino acid sequence consisting of 13 to 17 amino acid residues.
- each X is independently any amino acid residue other than cysteine, C is a cysteine residue; A is an alanine residue, H is a histidine residue; Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof, G is a glycine residue; E is a glutamic acid residue, L is a leucine residue; V is a valine residue, and W is a tryptophan residue. ) And an amino acid sequence consisting of 13 to 17 amino acid residues.
- each X is independently any amino acid residue other than cysteine, C is a cysteine residue; H is a histidine residue; Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof, G is a glycine residue; Xaa2 is a glutamic acid residue or an asparagine residue, L is a leucine residue; V is a valine residue; W is a tryptophan residue; Xaa3 is an alanine residue, a serine residue or a threonine residue, and Xaa4 is a tyrosine residue or a tryptophan residue
- the first and second amino acids and the 16th and 17th amino acid residues X from the N-terminus are missing when the number is 17 amino acid residues.
- Such peptides may be 13 amino acids long.
- “when 17 amino acid residues are used” means that when the amino acid residues of a peptide are referred to by amino acid number, from the N-terminal of 17 residues, which is the longest amino acid length for the peptide of formula I It is a term expressed for the sake of convenience in order to number from 1st to 17th in order.
- the peptide represented by Formula III is (X 1-3 ) -CAYH- (Xaa1) -GELVWC- (X 1-3 ) (III)
- each X is independently any amino acid residue other than cysteine, C is a cysteine residue; A is an alanine residue, Y is a tyrosine residue; H is a histidine residue; Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof, G is a glycine residue; E is a glutamic acid residue, L is a leucine residue; V is a valine residue, and W is a tryptophan residue. ) And an amino acid sequence consisting of 13 to 17 amino acid residues.
- amino acid sequence of the peptide of formula III above when it is 17 amino acid residues, the first and second amino acids from the N-terminal, and the 16th and 17th amino acid residues X may be deleted, Such a peptide may consist of 13 amino acids in length.
- amino acid residues other than cysteine (C) in the amino acid sequence of the peptides of the above formulas that is, amino acids 1 to 3, 5, 6, and 15 to 17 from the N-terminal in the case of 17 amino acid residues
- the peptide represented by Formula IV is DC- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -LVWCT (IV)
- D is an aspartic acid residue
- C is a cysteine residue
- H is a histidine residue
- Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof
- G is a glycine residue
- Xaa2 is a glutamic acid residue or an asparagine residue
- L is a leucine residue
- V is a valine residue
- W is a tryptophan residue
- T is a threonine residue
- Xaa3 is an alanine residue or a threonine residue
- Xaa4 is a tyrosine residue or a tryptophan residue.
- an amino acid sequence consisting of 13
- peptides of formula I are listed below in 1) to 18), but of course are not limited to these: 1) DCAYH (Xaa1) GELVWCT (SEQ ID NO: 1), 2) GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2), 3) RCAYH (Xaa1) GELVWCS (SEQ ID NO: 3), 4) GPRCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 4), 5) SPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 5), 6) GDDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 6), 7) GPSCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 7), 8) GPDCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 8), 9) GPDCAYH (Xaa1) GELVWCTHH (SEQ ID NO: 9), 10)
- the IgG-binding peptide described herein has the following formula V: DC- (Xaa2)-(Xaa3)-(Xaa4)-(Xaa1) -G- (Xaa5) -L- (Xaa6) -WCT (V) (Where D is an aspartic acid residue; C is a cysteine residue; G is a glycine residue; L is a leucine residue; W is a tryptophan residue; T is a threonine residue; Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof, Xaa2 is an alanine residue, a serine residue or a threonine residue, Xaa3 is a tryptophan residue or a tyrosine residue, Xaa4 is a histidine residue, arginine residue, serine residue or threonine residue, X
- linkers selected from the group consisting of linkers represented by:
- Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof, preferably an arginine residue, a lysine residue or A derivative thereof, a leucine residue or an asparagine residue, more preferably an arginine residue, a lysine residue or a derivative thereof, or a leucine residue.
- the type of the derivative is not particularly limited, but an acylated derivative such as an acetyl group or a propynyl group (the acylated derivative is represented by the general formula: R—CO—, in which R is a hydrocarbon, preferably Is an alkyl group having 1 to 6 carbon atoms.
- R is a hydrocarbon, preferably Is an alkyl group having 1 to 6 carbon atoms.
- derivatives include derivatives in which the ⁇ -amino group of a lysine residue is acylated, for example, acetylated.
- the IgG binding peptides described herein have at least two cysteine (C) residues spaced within each amino acid sequence, and the sulfide group of the cysteine residues has the following formula: It is characterized by being linked by a linker selected from the group consisting of linkers represented by
- the linker in the IgG-binding peptide is preferably A linker represented by It is a linker represented by these.
- R in the linker in the IgG-binding peptide is a substituted or unsubstituted alkyl, preferably a substituted or unsubstituted C1-C6 alkyl, that is, methyl group, ethyl group, propyl group, butyl group, pentyl Group or hexyl group.
- the substituent of R is not particularly limited, and examples thereof include a hydroxy group, a (mono or poly) ethylene oxide group, a carboxyl group, an alkoxy group, an acyl group, an alkyl group, an amide group, an ester group, a halogen group (F, Cl, Br, Or I), or a combination thereof.
- a wavy line means a bond part with a sulfide group.
- the linker is more excellent in stability, for example, resistance to alkali or reduction reaction, preferably alkali resistance than a normal disulfide bond.
- the method for preparing the peptide having the linker is not particularly limited.
- the following formula For example, the peptide having a linker represented by the following method: A step of mixing a peptide containing two cysteine residues and a compound having a reactive functional group (for example, a halogen group, an imidazole group, etc.) involved in a crosslinking reaction in the wavy portion of the linker, for example, under acidic conditions It can be obtained by the method of including.
- the halogen group is preferably selected from the group consisting of F, Cl, Br, and I, more preferably Cl, Br, and I.
- the halogen groups are preferably the same, more preferably all halogen groups are Cl.
- the conditions of the mixing step in this method are not particularly limited as long as a ligation reaction occurs between cysteine residues of the peptide.
- the reaction can be performed by mixing the peptide and the compound in an appropriate buffer at room temperature (eg, about 15 ° C. to 30 ° C.) or at a low temperature.
- the mixing step may be performed by adding a suitable amount of a base (or alkali) that promotes the ligation reaction, such as weakly basic inorganic or organic (for example, guanidinium chloride, sodium bicarbonate, and diethylamine).
- the mixing ratio of peptide and compound in the mixing step of this method is not particularly limited.
- the molar ratio of peptide to compound can be, for example, 1: 0.2 to 1:10, preferably 1: 0.5 to 1: 5 or 1: 1 to 1: 2.
- the mixing time (reaction time) in the mixing step is not limited as long as a ligation reaction occurs between cysteine residues of the peptide, but for example, 1 minute to 5 hours, preferably 10 minutes to 2 hours or 15 minutes to It can be 1 hour.
- the method further includes the step of separating impurities, for example, unreacted peptides and compounds from the mixture after performing the above steps, and purifying the peptide having a cysteine residue linked thereto, if necessary. Good.
- This step can be performed by a method known in the art, for example, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, chromatography such as HPLC, and the like.
- IgG-binding peptide described in the present specification may be modified by, for example, N-terminal PEGylation (polyethylene glycol addition), C-terminal amidation, or the like in order to improve its stability.
- the number of PEG molecules for PEGylation is not particularly limited, and for example, 1 to 50 molecules, 1 to 20 molecules, 2 to 10 molecules, 2 to 6 molecules, or 4 molecules of PEG can be added.
- the IgG-binding peptide described in the present specification may be multimerized.
- “multimerization” of an IgG-binding peptide means that two or more molecules of the above-mentioned IgG-binding peptide are linked via a covalent bond. It may be a hexamer, a 2-5 mer, a 2-4 mer, a 2-3 mer, preferably a dimer.
- the multimer of the peptide may have a spacer between the peptides. Multimerization can be performed by a method known to those skilled in the art, for example, by linking two or more molecules of the N-terminal amino group of the IgG-binding peptide via a spacer.
- the type of spacer is not particularly limited. For example, amino acids such as aspartic acid and glutamic acid having carboxyl groups at both ends, and functional groups such as carboxyl group, aldehyde group, epoxy group, N-hydroxysuccinimidyl group at both ends And polyalkylene oxide derivatives substituted with
- the IgG-binding peptides described herein have a binding affinity for human IgG of about 10 times or more, preferably about 50 times or more, more preferably compared to other human immunoglobulins (IgA, IgE, IgM). Can be about 200 times higher.
- the dissociation constant (Kd) for binding of the peptides described herein to human IgG can be determined by surface plasmon resonance spectral analysis (eg, using the BIACORE system), eg, less than 1 ⁇ 10 ⁇ 1 M, 1 ⁇ 10 Less than ⁇ 3 M, preferably less than 1 ⁇ 10 ⁇ 4 M, more preferably less than 1 ⁇ 10 ⁇ 5 M.
- the IgG binding peptides described herein can bind to the Fc domain of IgG.
- Peptides described in the present specification include conventional liquid phase synthesis methods, peptide synthesis methods such as solid phase synthesis methods, peptide synthesis using an automatic peptide synthesizer, etc.
- peptide synthesis methods such as solid phase synthesis methods, peptide synthesis using an automatic peptide synthesizer, etc.
- the peptide may be produced by a gene recombination method using a nucleic acid encoding the peptide described herein or a phage display method.
- a target peptide can be produced by incorporating a DNA encoding the amino acid sequence of the peptide described herein into an expression vector, introducing it into a host cell and culturing.
- the produced peptide can be obtained by conventional methods, for example, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, HPLC chromatography, ammonium sulfate fractionation, ultrafiltration, and immunoadsorption. It can be recovered or purified by a method or the like.
- amino acids prepared by protecting functional groups other than the ⁇ -amino group and ⁇ -carboxyl group to be bound of each amino acid are prepared.
- a peptide bond forming reaction is carried out between the ⁇ -amino group and ⁇ -carboxyl group of each amino acid.
- the carboxyl group of the amino acid residue located at the C-terminus of the peptide is bound to the solid phase via an appropriate spacer or linker.
- the amino-terminal protecting group of the dipeptide thus obtained is selectively removed, and a peptide bond is formed with the ⁇ -carboxyl group of the next amino acid.
- Production by a genetic recombination method is performed, for example, by inserting a DNA encoding the peptide described herein into an appropriate expression vector, introducing the vector into an appropriate host cell, culturing the cell, This can be done by a method comprising recovering the peptide of interest from the extracellular fluid.
- the vector is not limited, and is, for example, a vector such as a plasmid, phage, cosmid, phagemid, and virus. Plasmid vectors include, but are not limited to, E.
- Phage vectors include, but are not limited to, T7 phage display vectors (T7Select10-3b, T7Select1-1b, T7Select1-2a, T7Select1-2b, T7Select1-2c, etc.
- ⁇ phage vectors (Charon4A, Charon21A , EMBL3, EMBL4, ⁇ gt10, ⁇ gt11, ⁇ ZAP, ⁇ ZAPII, etc.).
- viral vectors include, but are not limited to, animal viruses such as retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, and Sendai viruses, and insect viruses such as baculoviruses.
- cosmid vectors include, but are not limited to, Lorist 6, Charomid 9-20, and Charomid 9-42.
- phagemid vectors include, but are not limited to, pSKAN, pBluescript, pBK, and pComb3H.
- the vector may include a regulatory sequence so that the target DNA can be expressed, a selection marker for selecting a vector containing the target DNA, a multicloning site for inserting the target DNA, and the like.
- regulatory sequences include promoters, enhancers, terminators, S-D sequences or ribosome binding sites, replication origins, poly A sites, and the like.
- an ampicillin resistance gene, a neomycin resistance gene, a kanamycin resistance gene, a dihydrofolate reductase gene, and the like can be used as the selection marker.
- Host cells for introducing the vector include bacteria such as Escherichia coli and Bacillus subtilis, yeast cells, insect cells, animal cells (for example, mammalian cells), plant cells, and the like, and transformation or transfer into these cells.
- the effects include, for example, calcium phosphate method, electroporation method, lipofection method, particle gun method, PEG method and the like.
- the transformed cells are cultured according to a usual method used for culturing host organisms. For example, a culture solution of microorganisms such as Escherichia coli and yeast cells contains a carbon source, a nitrogen source, and inorganic salts that can be assimilated by the host microorganism.
- the peptide produced by expression out of the cell is preferable to secrete the peptide produced by expression out of the cell. This can be performed by binding DNA encoding a peptide sequence that allows secretion of the peptide from the cell to the 5 ′ end of the DNA encoding the target peptide.
- the fusion peptide transferred to the cell membrane is cleaved by signal peptidase, and the target peptide is secreted and released into the medium.
- the target peptide accumulated in the cells can be recovered. In this case, the cells are physically or chemically destroyed, and the target peptide is recovered using protein purification techniques.
- the present invention relates to a column for separating IgG, preferably human IgG, comprising a solid phase carrier comprising the above-mentioned IgG-binding peptide.
- the column for IgG separation includes a column such as a chromatography column and a high performance liquid chromatography (HPLC) column for purification or separation of IgG.
- the size of the column is not particularly limited, and can be changed according to the use for analysis, purification, fractionation, etc., the amount to be applied (loaded) or injected, the length or inner diameter of the column, and the like.
- the column material may be a metal, plastic, glass, or the like that is normally used.
- the above-mentioned column can be produced by densely packing the above-described solid phase carrier of the present invention (which may be in a dry or wet state) into the column.
- the present invention relates to a kit for purifying IgG, preferably human IgG, comprising a solid phase carrier containing the above-mentioned IgG-binding peptide.
- the kit of the present invention may contain at least one of an instruction manual describing the analysis procedure and purification procedure of human IgG, a reagent and buffer necessary for purification, and a column for packing a solid phase carrier.
- the present invention includes the step of binding IgG to the solid phase carrier or the column for separating IgG, and the step of eluting the bound IgG and recovering IgG, preferably purification of human IgG Regarding the method.
- the binding step can be performed by a method known to those skilled in the art. For example, equilibrate with the above-mentioned solid phase carrier or IgG separation column with an appropriate buffer, and apply a solution containing IgG at a low temperature of 0 ° C. to room temperature, preferably 0 ° C. to about 10 ° C., more preferably about 4 ° C., IgG is bound to the peptide on the solid support.
- a binding step can be performed by applying to a column using a buffer having a neutral pH, for example, pH 6.0 to 7.5.
- the elution step can also be performed by methods known to those skilled in the art.
- an acidic pH range for example, pH 2-4 buffer (for example, pH 3.5 to pH 2.5 0.2 M glycine-HCl buffer or 20 mM mM citrate buffer containing 0.3 M NaCl) is applied to the column.
- it may be eluted by competitive elution using the above-mentioned IgG-binding peptide.
- elution with an acid is preferable from the viewpoint of cost.
- the solid phase carrier or column is washed with an alkaline solution such as sodium hydroxide solution, potassium hydroxide solution, and potassium hydroxide solution (for example, 0.1 M sodium hydroxide solution).
- the method of the present invention may optionally include the step of regenerating the solid support or column by washing with an alkaline solution.
- IgG Whether or not IgG has been recovered can be determined by, for example, confirmation of molecular weight by electrophoresis and optionally subsequent Western blotting using an anti-IgG antibody.
- electrophoresis may be performed by SDS-PAGE using a 5-20% acrylamide gradient gel
- Western blot is performed by transferring the protein after electrophoresis to a PVDF membrane, blocking with skim milk, and then anti-IgG ⁇ chain goat.
- Detection can be performed with an antibody and an HRP-labeled anti-goat IgG mouse antibody.
- the method of the present invention is useful for obtaining an IgG-rich fraction in the step of purifying IgG from an IgG-containing product produced by various methods. Therefore, it is preferable to use the method of the present invention in column chromatography such as affinity chromatography and HPLC.
- column chromatography such as affinity chromatography and HPLC.
- conventional protein purification techniques such as gel filtration chromatography, ion exchange column chromatography, reverse phase column chromatography, etc., ammonium sulfate fractionation, Ultrafiltration or the like can be combined as appropriate.
- Example 1 Preparation of IgG-binding peptide and measurement of binding affinity
- the following 6 IgG-binding peptides whose N-termini were blocked with biotinylated PEG4, were synthesized according to a conventional method by Fmoc solid phase synthesis: DCAYH (Xaa1) GELVWCT (SEQ ID NO: 1, where Xaa1 is arginine and the C-terminal is amidated); GPRCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 4, where Xaa1 is arginine and the C-terminus is amidated); GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2, where Xaa1 is arginine and the C-terminal is amidated); GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2, where Xaa1 is leucine and C-terminal is amidated); GPDCAYH (Xaa1)
- intramolecular SS bonds were formed under oxidizing conditions in aqueous solution at pH 8.5, and using reverse phase HPLC, the flow rate was 1.0 ml / min, 10% to 60% containing 0.1% TFA.
- Peptides with intramolecular SS bonds were purified by gradient elution of acetonitrile.
- the affinity analysis of the purified IgG-binding peptide was performed by the following method.
- 0.4MCMEDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and 0.1M onto CM5 sensor chip set in BIAcoreT200 (GE healthcare)
- sulfo-NHS sulfo-N-hydroxysuccinimide
- the sensor chip is activated by injecting into the sensor chip for 7 minutes at a flow rate of 10 ⁇ l / ml, pH 4.0 IgG was immobilized so that the amount of immobilization was 4000 to 5000 in terms of RU value under the condition of (10 mM mM acetic acid Na).
- HBS-EP buffer 0.01 M HEPES, 0.15 M NaCl, 0.005% Tween 20, 3 mM mM EDTA, pH 7.0
- Example 2 Preparation of IgG-binding peptide cross-linked by linker
- NH2-PEG4 synthetic peptide GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2, Xaa1 is arginine, C-terminal is amidated) is synthesized on Fmoc solid phase synthesis on peptide synthetic beads (Rink-amide-Chemmatrix resin, Biotage) The compound was synthesized according to a conventional method.
- the peptide was obtained after excision and deprotection of the peptide from the resin.
- -Propanone (2.9 mg, 23.4 mol, 1.5 equimolar) was added and stirred at room temperature. After 1 hour, the completion of the reaction was confirmed by HPLC analysis, and the reaction solution was directly purified by HPLC to obtain a cyclized peptide (33 mg, 7.8 mg, yield 50%).
- 1.1 mg of the peptide (Fmoc-HN-PEG4-GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2, Xaa1 is arginine, C-terminal is amidated)) prepared in the same manner as above is dissolved in 220 ⁇ L of DMF. (2 mM), and 22 ⁇ L (0.5 equivalent mole) of 10 mM thiocarbonyldiimidazole dissolved in acetonitrile was added thereto, followed by stirring on ice for 2.5 hours.
- Example 3 Separation of ⁇ -globulin derived from human serum
- dichloroacetone cross-linked peptide can be used as a human antibody purified affinity ligand was immobilized on an NHS-activated prepacked column (GE Healthcare) and subjected to various evaluations.
- the peptide immobilization column was prepared by the following method. A syringe was used for feeding the solution.
- the prepared peptide-immobilized column was connected to a liquid chromatography device AKTAexplore® (GE® Healthcare) and equilibrated with an adsorption solution.
- AKTAexplore® GE® Healthcare
- 1 mg / mL human serum-derived ⁇ -globulin (Wako) dissolved in the adsorption solution was fed at a flow rate of 1 mg / min for 1 mg / min.
- the column was washed with the adsorption solution, and the adsorbed component was eluted by feeding an acidic elution solution (20 mM mM citric acid, pH 2.5). Protein elution from the column was detected by absorbance at 280 nm.
- the experimental results are shown in FIG.
- Example 2 As shown in FIG. 1, elution of human serum-derived ⁇ -globulin adsorbed on the column was confirmed due to a decrease in pH, and thus it was clear that the peptide prepared in Example 2 could be used as an affinity column ligand. became.
- Example 4 Competitive elution with peptide
- 1 mg of human-derived serum ⁇ -globulin was fed to the column prepared in the same manner as in Example 3 and adsorbed. After washing the column with an adsorption solution, 2.5 mL of 0.4 mg / mL peptide a solution prepared in Example 2 dissolved in the adsorption solution was fed. Each fraction fractioned with 0.5 mL was subjected to SDS-PAGE under reducing conditions according to a conventional method. Protein detection was performed by CBB staining. For comparison, the same operation was performed for citric acid elution.
- Example 5 Dynamic binding capacity (DBC) measurement
- DBD Dynamic binding capacity
- Example 6 Evaluation of alkali resistance
- 5 mL of a 0.1 M sodium hydroxide solution was fed to a 1 mg peptide-immobilized 1 mL column prepared in the same manner as in Example 3. Thereafter, the adsorbed solution was washed, and DBC measurement was performed at a flow rate of 1 mL / min in the same manner as in Example 5. Subsequently, alkali resistance was evaluated by repeating sodium hydroxide solution treatment / DBC measurement 5 times. The DBC fluctuation rate was determined by setting DBC before sodium hydroxide treatment to 100%.
- Comparative Example 1 As Comparative Example 1, a column was prepared by immobilizing 1 mg of a peptide crosslinked with a disulfide bond, and alkali resistance was evaluated in the same manner as in Example 5. The results of Example 5 and Comparative Example 1 are shown in FIG. 4, and the measured values are summarized in Table 3.
- the peptide crosslinked with a disulfide bond had a DBC lowered to 86.4% after 5 treatments with sodium hydroxide (Comparative Example 1).
- the dichloroacetone cross-linked peptide was found to have high alkali resistance without a decrease in DBC.
- the stability of the peptide contained in the solid phase carrier of the present invention is improved by crosslinking the sulfide group in the cysteine residue with a linker having a specific structure. Therefore, the solid phase carrier of the present invention can be used for efficient IgG purification because the IgG binding ability is difficult to be reduced by an alkali washing step or the like, and can be used for efficient production of IgG used as a pharmaceutical product. Can do.
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Abstract
Description
(1)ヒトIgGと結合可能であることを特徴とするペプチドを固定化した固相担体であって、
前記ペプチドが、下記の式I:
(X1-3)-C-(X2)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(X1-3) (I)
(式中、Xの各々は独立的にシステイン以外の任意のアミノ酸残基であり、
Cはシステイン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Xaa2はグルタミン酸残基又はアスパラギン残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、かつ
Wはトリプトファン残基である。)
によって表される、13~17アミノ酸残基からなるアミノ酸配列を含み、かつ前記ペプチドの外側の2つのシステイン残基中のスルフィド基が、以下の式:
(2)前記ペプチドが、下記の式II:
(X1-3)-C-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(X1-3) (II)
(式中、Xの各々は独立的にシステイン以外の任意のアミノ酸残基であり、
Cはシステイン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Xaa2はグルタミン酸残基又はアスパラギン残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、
Wはトリプトファン残基であり、
Xaa3はアラニン残基、セリン残基又はトレオニン残基であり、かつ
Xaa4はチロシン残基又はトリプトファン残基である。)
によって表される、13~17アミノ酸残基からなるアミノ酸配列を含む、(1)に記載の固相担体。
(3)前記ペプチドが、下記の式III:
(X1-3)-C-A-Y-H-(Xaa1)-G-E-L-V-W-C-(X1-3) (III)
(式中、Xの各々は独立的にシステイン以外の任意のアミノ酸残基であり、
Cはシステイン残基であり、
Aはアラニン残基であり、
Yはチロシン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Eはグルタミン酸残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、かつ
Wはトリプトファン残基である。)
によって表される、13~17アミノ酸残基からなるアミノ酸配列を含む、(1)又は(2)に記載の固相担体。
(4)17アミノ酸残基とした場合の、前記ペプチドのN末端から1~3、15~17番目の各アミノ酸残基が、
1番目のアミノ酸残基= S、G、F又は、なし
2番目のアミノ酸残基= D、G、A、S、P、ホモシステイン、又は、なし
3番目のアミノ酸残基= S、D、T、N、E又はR、
15番目のアミノ酸残基= S、T又はD、
16番目のアミノ酸残基= H、G、Y、T、N、D、F、ホモシステイン、又は、なし、
17番目のアミノ酸残基= Y、F、H、M又は、なし
である、(1)~(3)のいずれかに記載の固相担体。
(5)前記ペプチドが、以下の1)~14)のいずれかのアミノ酸配列からなる、ただし、Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、Xaa2はホモシステインである、(4)に記載の固相担体:
1)DCAYH(Xaa1)GELVWCT(配列番号1)
2)GPDCAYH(Xaa1)GELVWCTFH(配列番号2)
3)RCAYH(Xaa1)GELVWCS(配列番号3)
4)GPRCAYH(Xaa1)GELVWCSFH(配列番号4)
5)SPDCAYH(Xaa1)GELVWCTFH(配列番号5)
6)GDDCAYH(Xaa1)GELVWCTFH(配列番号6)
7)GPSCAYH(Xaa1)GELVWCTFH(配列番号7)
8)GPDCAYH(Xaa1)GELVWCSFH(配列番号8)
9)GPDCAYH(Xaa1)GELVWCTHH(配列番号9)
10)GPDCAYH(Xaa1)GELVWCTFY(配列番号10)
11)SPDCAYH(Xaa1)GELVWCTFY(配列番号11)
12)SDDCAYH(Xaa1)GELVWCTFY(配列番号12)
13)RGNCAYH(Xaa1)GQLVWCTYH(配列番号13)
14)G(Xaa2)DCAYH(Xaa1)GELVWCT(Xaa2)H(配列番号14)。
(6)前記ペプチドが、下記の式IV:
D-C-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-T (IV)
(式中、
Dはアスパラギン酸残基であり、
Cはシステイン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Xaa2はグルタミン酸残基又はアスパラギン残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、
Wはトリプトファン残基であり、
Tはトレオニン残基であり、
Xaa3はアラニン残基又はトレオニン残基であり、かつ、
Xaa4はチロシン残基又はトリプトファン残基である。)によって表される、13アミノ酸残基からなるアミノ酸配列を含む、(1)又は(2)に記載の固相担体。
(7)前記ペプチドが、以下の1)~4)のいずれかのアミノ酸配列からなる、ただし、Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体である、(6)に記載の固相担体:
1)DCTYH(Xaa1)GNLVWCT(配列番号15)
2)DCAYH(Xaa1)GNLVWCT(配列番号16)
3)DCTYH(Xaa1)GELVWCT(配列番号17)
4)DCAWH(Xaa1)GELVWCT(配列番号18)。 Accordingly, the present invention includes the following aspects.
(1) A solid phase carrier immobilizing a peptide characterized by being capable of binding to human IgG,
Said peptide is represented by the following formula I:
(X 1-3 ) -C- (X 2 ) -H- (Xaa1) -G- (Xaa2) -LVWC- (X 1-3 ) (I)
(In the formula, each X is independently any amino acid residue other than cysteine,
C is a cysteine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
Xaa2 is a glutamic acid residue or an asparagine residue,
L is a leucine residue;
V is a valine residue, and
W is a tryptophan residue. )
The sulfide group in the two cysteine residues outside the peptide comprising an amino acid sequence consisting of 13-17 amino acid residues represented by
(2) The peptide is represented by the following formula II:
(X 1-3 ) -C- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -LVWC- (X 1-3 ) (II)
(In the formula, each X is independently any amino acid residue other than cysteine,
C is a cysteine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
Xaa2 is a glutamic acid residue or an asparagine residue,
L is a leucine residue;
V is a valine residue;
W is a tryptophan residue;
Xaa3 is an alanine residue, a serine residue or a threonine residue, and
Xaa4 is a tyrosine residue or a tryptophan residue. )
The solid phase carrier according to (1), comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by:
(3) The peptide is represented by the following formula III:
(X 1-3 ) -CAYH- (Xaa1) -GELVWC- (X 1-3 ) (III)
(In the formula, each X is independently any amino acid residue other than cysteine,
C is a cysteine residue;
A is an alanine residue,
Y is a tyrosine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
E is a glutamic acid residue,
L is a leucine residue;
V is a valine residue, and
W is a tryptophan residue. )
The solid phase carrier according to (1) or (2), comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by:
(4) In the case of 17 amino acid residues, the 1st to 3rd and 15th to 17th amino acid residues from the N-terminus of the peptide are
1st amino acid residue = S, G, F or none
2nd amino acid residue = D, G, A, S, P, homocysteine or none
3rd amino acid residue = S, D, T, N, E or R,
15th amino acid residue = S, T or D,
16th amino acid residue = H, G, Y, T, N, D, F, homocysteine or none,
The solid phase carrier according to any one of (1) to (3), wherein the 17th amino acid residue is Y, F, H, M, or none.
(5) The peptide is composed of any one of the following amino acid sequences 1) to 14), wherein Xaa1 is an arginine residue, a lysine residue, a leucine residue, an asparagine residue, or a derivative thereof: , Xaa2 is homocysteine, the solid phase support according to (4):
1) DCAYH (Xaa1) GELVWCT (SEQ ID NO: 1)
2) GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2)
3) RCAYH (Xaa1) GELVWCS (SEQ ID NO: 3)
4) GPRCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 4)
5) SPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 5)
6) GDDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 6)
7) GPSCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 7)
8) GPDCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 8)
9) GPDCAYH (Xaa1) GELVWCTHH (SEQ ID NO: 9)
10) GPDCAYH (Xaa1) GELVWCTFY (SEQ ID NO: 10)
11) SPDCAYH (Xaa1) GELVWCTFY (SEQ ID NO: 11)
12) SDDCAYH (Xaa1) GELVWCTFY (SEQ ID NO: 12)
13) RGNCAYH (Xaa1) GQLVWCTYH (SEQ ID NO: 13)
14) G (Xaa2) DCAYH (Xaa1) GELVWCT (Xaa2) H (SEQ ID NO: 14).
(6) The peptide is represented by the following formula IV:
DC- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -LVWCT (IV)
(Where
D is an aspartic acid residue;
C is a cysteine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
Xaa2 is a glutamic acid residue or an asparagine residue,
L is a leucine residue;
V is a valine residue;
W is a tryptophan residue;
T is a threonine residue;
Xaa3 is an alanine residue or a threonine residue, and
Xaa4 is a tyrosine residue or a tryptophan residue. The solid phase carrier according to (1) or (2), comprising an amino acid sequence consisting of 13 amino acid residues represented by
(7) The peptide is composed of any one of the following amino acid sequences 1) to 4), wherein Xaa1 is an arginine residue, a lysine residue, a leucine residue, an asparagine residue, or a derivative thereof: , (6) solid phase carrier:
1) DCTYH (Xaa1) GNLVWCT (SEQ ID NO: 15)
2) DCAYH (Xaa1) GNLVWCT (SEQ ID NO: 16)
3) DCTYH (Xaa1) GELVWCT (SEQ ID NO: 17)
4) DCAWH (Xaa1) GELVWCT (SEQ ID NO: 18).
前記ペプチドが、下記の式V:
D-C-(Xaa2)-(Xaa3)-(Xaa4)-(Xaa1)-G-(Xaa5)-L-(Xaa6)-W-C-T (V)
(式中、
Dはアスパラギン酸残基であり、
Cはシステイン残基であり、
Gはグリシン残基であり、
Lはロイシン残基であり、
Wはトリプトファン残基であり、
Tはトレオニン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Xaa2はアラニン残基、セリン残基又はトレオニン残基であり、
Xaa3はトリプトファン残基又はチロシン残基であり、
Xaa4はヒスチジン残基、アルギニン残基、セリン残基又はトレオニン残基であり、
Xaa5はグルタミン酸残基、アスパラギン残基、アルギニン残基、又はアスパラギン酸残基であり、かつ
Xaa6はイソロイシン残基又はバリン残基である)によって表される、13アミノ酸残基からなるアミノ酸配列を含み、かつ前記ペプチドの外側の2つのシステイン残基中のスルフィド基が、以下の式:
(9)Xaa1がアルギニン残基、リシン残基若しくはそのアシル化誘導体、又はロイシン残基である、(1)~(8)のいずれかに記載の固相担体。
(10)前記ペプチドが、以下のアミノ酸配列からなる、(1)に記載の固相担体:
GPDCAYHRGELVWCTFH(配列番号31)。
(11)前記リンカーが、以下の式:
(12)前記ペプチドのN末端がPEG化されている、(1)~(11)のいずれかに記載の固相担体。
(13)前記ペプチドのC末端がアミド化されている、(1)~(12)のいずれかに記載の固相担体。
(14)前記ペプチドが多量体化されている、(1)~(13)のいずれかに記載の固相担体。
(15)前記ペプチドの多量体が、該ペプチド間にスペーサーを有する、(14)に記載の固相担体。
(16)前記ペプチドと固相間にスペーサーを有する、(1)~(15)のいずれかに記載の固相担体。
(17)(1)~(16)のいずれかに記載の固相担体を含む、IgG分離用カラム。
(18)(1)~(16)のいずれかに記載の固相担体又は(17)に記載のIgG分離用カラムを含む、IgGの精製のためのキット。
(19)(1)~(16)のいずれかに記載の固相担体、又は(17)に記載のIgG分離用カラムにIgGを結合させる工程、及び
結合したIgGを溶出させてIgGを回収する工程を含む、IgGの精製方法。 (8) A solid phase carrier immobilizing a peptide that is capable of binding to human IgG,
Said peptide is represented by the following formula V:
DC- (Xaa2)-(Xaa3)-(Xaa4)-(Xaa1) -G- (Xaa5) -L- (Xaa6) -WCT (V)
(Where
D is an aspartic acid residue;
C is a cysteine residue;
G is a glycine residue;
L is a leucine residue;
W is a tryptophan residue;
T is a threonine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
Xaa2 is an alanine residue, a serine residue or a threonine residue,
Xaa3 is a tryptophan residue or a tyrosine residue,
Xaa4 is a histidine residue, arginine residue, serine residue or threonine residue,
Xaa5 is a glutamic acid residue, an asparagine residue, an arginine residue, or an aspartic acid residue; and
Xaa6 is an isoleucine residue or a valine residue), and the sulfide group in the two cysteine residues outside the peptide is represented by the following formula:
(9) The solid phase carrier according to any one of (1) to (8), wherein Xaa1 is an arginine residue, a lysine residue or an acylated derivative thereof, or a leucine residue.
(10) The solid phase carrier according to (1), wherein the peptide has the following amino acid sequence:
GPDCAYHRGELVWCTFH (SEQ ID NO: 31).
(11) The linker is represented by the following formula:
(12) The solid phase carrier according to any one of (1) to (11), wherein the N-terminus of the peptide is PEGylated.
(13) The solid phase carrier according to any one of (1) to (12), wherein the C-terminus of the peptide is amidated.
(14) The solid phase carrier according to any one of (1) to (13), wherein the peptide is multimerized.
(15) The solid phase carrier according to (14), wherein the multimer of the peptide has a spacer between the peptides.
(16) The solid phase carrier according to any one of (1) to (15), which has a spacer between the peptide and the solid phase.
(17) An IgG separation column comprising the solid phase carrier according to any one of (1) to (16).
(18) A kit for purifying IgG comprising the solid phase carrier according to any one of (1) to (16) or the IgG separation column according to (17).
(19) The step of binding IgG to the solid phase carrier according to any one of (1) to (16) or the IgG separation column according to (17), and recovering IgG by eluting the bound IgG A method for purifying IgG, comprising a step.
一態様において、本発明は、IgG結合ペプチドを含む固相担体に関する。本明細書における「固相担体」としては、限定するものではないが、ガラスビーズ、シリカゲル等の無機担体、架橋ポリビニルアルコール、架橋ポリアクリレート、架橋ポリアクリルアミド、架橋ポリスチレン等の合成高分子や結晶性セルロース、架橋セルロース、架橋アガロース、架橋デキストラン等の多糖類からなる有機担体、さらにはこれらの組み合わせによって得られる有機-有機、有機-無機等の複合担体等が挙げられるが、中でも親水性担体は非特異吸着が比較的少なく、IgG結合ペプチドの選択性が良好であるため好ましい。ここでいう親水性担体とは、担体を構成する化合物を平板状にしたときの水との接触角が60度以下の担体を示す。この様な担体としてはセルロース、キトサン、デキストラン等の多糖類、ポリビニルアルコール、エチレン-酢酸ビニル共重合体けん化物、ポリアクリルアミド、ポリアクリル酸、ポリメタクリル酸、ポリメタクリル酸メチル、ポリアクリル酸グラフト化ポリエチレン、ポリアクリルアミドグラフト化ポリエチレン、ガラス等からなる担体が代表例として挙げられる。 <Solid phase carrier containing IgG-binding peptide>
In one aspect, the invention relates to a solid support comprising an IgG binding peptide. The “solid phase carrier” in the present specification is not limited, but includes inorganic carriers such as glass beads and silica gel, synthetic polymers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, and crosslinked polystyrene, and crystallinity. Examples include organic carriers composed of polysaccharides such as cellulose, crosslinked cellulose, crosslinked agarose, and crosslinked dextran, and organic-organic, organic-inorganic composite carriers obtained by a combination thereof, among which hydrophilic carriers are not. It is preferable because specific adsorption is relatively small and selectivity of an IgG-binding peptide is good. The term “hydrophilic carrier” as used herein refers to a carrier having a contact angle with water of 60 ° or less when the compound constituting the carrier is formed into a flat plate shape. Such carriers include polysaccharides such as cellulose, chitosan, dextran, polyvinyl alcohol, saponified ethylene-vinyl acetate copolymer, polyacrylamide, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylic acid grafting Representative examples include carriers made of polyethylene, polyacrylamide grafted polyethylene, glass and the like.
本明細書中で使用する「IgG」は、哺乳動物、例えばヒト及びチンパンジー等の霊長類、ラット、マウス、及びウサギ等の実験動物、ブタ、ウシ、ウマ、ヒツジ、及びヤギ等の家畜動物、並びにイヌ及びネコ等の愛玩動物のIgG、好ましくはヒトのIgG(IgG1、IgG2、IgG3又はIgG4)を指すものとする。本明細書におけるIgGは、さらに好ましくは、ヒトIgG1、IgG2、若しくはIgG4、又はウサギIgGであり、特に好ましくはヒトIgG1、IgG2、又はIgG4である。 The IgG-binding peptide that can be contained in the solid phase carrier of the present invention is described in detail below.
As used herein, “IgG” refers to mammals such as primates such as humans and chimpanzees, laboratory animals such as rats, mice, and rabbits, and domestic animals such as pigs, cows, horses, sheep, and goats, In addition, it refers to IgG of pet animals such as dogs and cats, preferably human IgG (IgG1, IgG2, IgG3 or IgG4). The IgG in the present specification is more preferably human IgG1, IgG2, or IgG4, or rabbit IgG, and particularly preferably human IgG1, IgG2, or IgG4.
(X1-3)-C-(X2)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(X1-3) (I)
(式中、Xの各々は独立的にシステイン以外の任意のアミノ酸残基であり、
Cはシステイン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Xaa2はグルタミン酸残基又はアスパラギン残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、かつ
Wはトリプトファン残基である。)
によって表される、13~17アミノ酸残基からなるアミノ酸配列を含み、かつ前記ペプチドの外側の2つのシステイン残基中のスルフィド基が、以下の式:
(X 1-3 ) -C- (X 2 ) -H- (Xaa1) -G- (Xaa2) -LVWC- (X 1-3 ) (I)
(In the formula, each X is independently any amino acid residue other than cysteine,
C is a cysteine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
Xaa2 is a glutamic acid residue or an asparagine residue,
L is a leucine residue;
V is a valine residue, and
W is a tryptophan residue. )
The sulfide group in the two cysteine residues outside the peptide comprising an amino acid sequence consisting of 13-17 amino acid residues represented by
(X1-3)-C-(X1)-Y-H-(Xaa1)-G-N-L-V-W-C-(X1-3) (I')
(式中、Xの各々は独立的にシステイン以外の任意のアミノ酸残基であり、
Cはシステイン残基であり、
Yはチロシン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Nはアスパラギン残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、かつ
Wはトリプトファン残基である。)
によって表される、13~17アミノ酸残基からなるアミノ酸配列を含む。 That is, the peptide represented by the formula I ′ is
(X 1-3 ) -C- (X 1 ) -YH- (Xaa1) -GNLVWC- (X 1-3 ) (I ')
(In the formula, each X is independently any amino acid residue other than cysteine,
C is a cysteine residue;
Y is a tyrosine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
N is an asparagine residue,
L is a leucine residue;
V is a valine residue, and
W is a tryptophan residue. )
And an amino acid sequence consisting of 13 to 17 amino acid residues.
(X1-3)-C-A-(X1)-H-(Xaa1)-G-E-L-V-W-C-(X1-3) (I'')
(式中、Xの各々は独立的にシステイン以外の任意のアミノ酸残基であり、
Cはシステイン残基であり、
Aはアラニン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Eはグルタミン酸残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、かつ
Wはトリプトファン残基である。)
によって表される、13~17アミノ酸残基からなるアミノ酸配列を含む。 The peptide represented by formula I ''
(X 1-3 ) -CA- (X 1 ) -H- (Xaa1) -GELVWC- (X 1-3 ) (I '')
(In the formula, each X is independently any amino acid residue other than cysteine,
C is a cysteine residue;
A is an alanine residue,
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
E is a glutamic acid residue,
L is a leucine residue;
V is a valine residue, and
W is a tryptophan residue. )
And an amino acid sequence consisting of 13 to 17 amino acid residues.
(X1-3)-C-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(X1-3) (II)
(式中、Xの各々は独立的にシステイン以外の任意のアミノ酸残基であり、
Cはシステイン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Xaa2はグルタミン酸残基又はアスパラギン残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、
Wはトリプトファン残基であり、
Xaa3はアラニン残基、セリン残基又はトレオニン残基であり、かつ
Xaa4はチロシン残基又はトリプトファン残基である。)
によって表される、13~17アミノ酸残基からなるアミノ酸配列を含む。 That is, the peptide represented by Formula II is
(X 1-3 ) -C- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -LVWC- (X 1-3 ) (II)
(In the formula, each X is independently any amino acid residue other than cysteine,
C is a cysteine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
Xaa2 is a glutamic acid residue or an asparagine residue,
L is a leucine residue;
V is a valine residue;
W is a tryptophan residue;
Xaa3 is an alanine residue, a serine residue or a threonine residue, and
Xaa4 is a tyrosine residue or a tryptophan residue. )
And an amino acid sequence consisting of 13 to 17 amino acid residues.
(X1-3)-C-A-Y-H-(Xaa1)-G-E-L-V-W-C-(X1-3) (III)
(式中、Xの各々は独立的にシステイン以外の任意のアミノ酸残基であり、
Cはシステイン残基であり、
Aはアラニン残基であり、
Yはチロシン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Eはグルタミン酸残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、かつ
Wはトリプトファン残基である。)
によって表される、13~17アミノ酸残基からなるアミノ酸配列を含む。 That is, the peptide represented by Formula III is
(X 1-3 ) -CAYH- (Xaa1) -GELVWC- (X 1-3 ) (III)
(In the formula, each X is independently any amino acid residue other than cysteine,
C is a cysteine residue;
A is an alanine residue,
Y is a tyrosine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
E is a glutamic acid residue,
L is a leucine residue;
V is a valine residue, and
W is a tryptophan residue. )
And an amino acid sequence consisting of 13 to 17 amino acid residues.
1番目のアミノ酸残基= S、G、F又は、なし
2番目のアミノ酸残基= D、G、A、S、P、ホモシステイン又は、なし
3番目のアミノ酸残基= S、D、T、N、E又はR、
15番目のアミノ酸残基= S、T又はD、
16番目のアミノ酸残基= H、G、Y、T、N、D、F、ホモシステイン又は、なし、
17番目のアミノ酸残基= Y、F、H、M又は、なし。
5番目のアミノ酸残基= A又はT、
6番目のアミノ酸残基= Y又はW。 Furthermore, amino acid residues other than cysteine (C) in the amino acid sequence of the peptides of the above formulas, that is,
1st amino acid residue = S, G, F or none
2nd amino acid residue = D, G, A, S, P, homocysteine or none
3rd amino acid residue = S, D, T, N, E or R,
15th amino acid residue = S, T or D,
16th amino acid residue = H, G, Y, T, N, D, F, homocysteine or none,
17th amino acid residue = Y, F, H, M or none.
5th amino acid residue = A or T,
6th amino acid residue = Y or W.
D-C-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-T (IV)
(式中、
Dはアスパラギン酸残基であり、
Cはシステイン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Xaa2はグルタミン酸残基又はアスパラギン残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、
Wはトリプトファン残基であり、
Tはトレオニン残基であり、
Xaa3はアラニン残基又はトレオニン残基であり、かつ、
Xaa4はチロシン残基又はトリプトファン残基である。)によって表される、13アミノ酸残基からなるアミノ酸配列を含む。 That is, the peptide represented by Formula IV is
DC- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -LVWCT (IV)
(Where
D is an aspartic acid residue;
C is a cysteine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
Xaa2 is a glutamic acid residue or an asparagine residue,
L is a leucine residue;
V is a valine residue;
W is a tryptophan residue;
T is a threonine residue;
Xaa3 is an alanine residue or a threonine residue, and
Xaa4 is a tyrosine residue or a tryptophan residue. And an amino acid sequence consisting of 13 amino acid residues.
1)DCAYH(Xaa1)GELVWCT(配列番号1)、
2)GPDCAYH(Xaa1)GELVWCTFH(配列番号2)、
3)RCAYH(Xaa1)GELVWCS(配列番号3)、
4)GPRCAYH(Xaa1)GELVWCSFH(配列番号4)、
5)SPDCAYH(Xaa1)GELVWCTFH(配列番号5)、
6)GDDCAYH(Xaa1)GELVWCTFH(配列番号6)、
7)GPSCAYH(Xaa1)GELVWCTFH(配列番号7)、
8)GPDCAYH(Xaa1)GELVWCSFH(配列番号8)、
9)GPDCAYH(Xaa1)GELVWCTHH(配列番号9)、
10)GPDCAYH(Xaa1)GELVWCTFY(配列番号10)、
11)SPDCAYH(Xaa1)GELVWCTFY(配列番号11)、
12)SDDCAYH(Xaa1)GELVWCTFY(配列番号12)、
13)RGNCAYH(Xaa1)GQLVWCTYH(配列番号13)、
14)G(Xaa2)DCAYH(Xaa1)GELVWCT(Xaa2)H(配列番号14)、
15)DCTYH(Xaa1)GNLVWCT(配列番号15)、
16)DCAYH(Xaa1)GNLVWCT(配列番号16)、
17)DCTYH(Xaa1)GELVWCT(配列番号17)、及び
18)DCAWH(Xaa1)GELVWCT(配列番号18)
(式中、Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、Xaa2はホモシステインであり、好ましくはホモシステイン同士は互いにジスルフィド結合を形成している)。 Some specific examples of peptides of formula I are listed below in 1) to 18), but of course are not limited to these:
1) DCAYH (Xaa1) GELVWCT (SEQ ID NO: 1),
2) GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2),
3) RCAYH (Xaa1) GELVWCS (SEQ ID NO: 3),
4) GPRCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 4),
5) SPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 5),
6) GDDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 6),
7) GPSCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 7),
8) GPDCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 8),
9) GPDCAYH (Xaa1) GELVWCTHH (SEQ ID NO: 9),
10) GPDCAYH (Xaa1) GELVWCTFY (SEQ ID NO: 10),
11) SPDCAYH (Xaa1) GELVWCTFY (SEQ ID NO: 11),
12) SDDCAYH (Xaa1) GELVWCTFY (SEQ ID NO: 12),
13) RGNCAYH (Xaa1) GQLVWCTYH (SEQ ID NO: 13),
14) G (Xaa2) DCAYH (Xaa1) GELVWCT (Xaa2) H (SEQ ID NO: 14),
15) DCTYH (Xaa1) GNLVWCT (SEQ ID NO: 15),
16) DCAYH (Xaa1) GNLVWCT (SEQ ID NO: 16),
17) DCTYH (Xaa1) GELVWCT (SEQ ID NO: 17) and 18) DCAWH (Xaa1) GELVWCT (SEQ ID NO: 18)
(Wherein Xaa1 is an arginine residue, lysine residue, leucine residue, or asparagine residue, or a derivative thereof, Xaa2 is a homocysteine, and preferably homocysteines form a disulfide bond with each other. )
1)DCAYH(Xaa1)GELVWCT(配列番号1、但し、Xaa1はR)、
2)GPDCAYH(Xaa1)GELVWCTFH(配列番号2、但し、Xaa1はR、L、K又はアセチル化リシン)、及び
4)GPRCAYH(Xaa1)GELVWCSFH(配列番号4、但し、Xaa1はR)、
が挙げられ、特に好ましい例として、GPDCAYHRGELVWCTFH(配列番号31)が挙げられる。 As a preferred embodiment of the peptide of formula I,
1) DCAYH (Xaa1) GELVWCT (SEQ ID NO: 1, where Xaa1 is R),
2) GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2, where Xaa1 is R, L, K or acetylated lysine), and 4) GPRCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 4, where Xaa1 is R),
A particularly preferred example is GPDCAYHRGELVWCTFH (SEQ ID NO: 31).
D-C-(Xaa2)-(Xaa3)-(Xaa4)-(Xaa1)-G-(Xaa5)-L-(Xaa6)-W-C-T (V)
(式中、
Dはアスパラギン酸残基であり、
Cはシステイン残基であり、
Gはグリシン残基であり、
Lはロイシン残基であり、
Wはトリプトファン残基であり、
Tはトレオニン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Xaa2はアラニン残基、セリン残基又はトレオニン残基であり、
Xaa3はトリプトファン残基又はチロシン残基であり、
Xaa4はヒスチジン残基、アルギニン残基、セリン残基又はトレオニン残基であり、
Xaa5はグルタミン酸残基、アスパラギン残基、アルギニン残基、又はアスパラギン酸残基であり、かつ
Xaa6はイソロイシン残基又はバリン残基である)によって表される、13アミノ酸残基からなるアミノ酸配列を含み、かつ前記ペプチドの外側の2つのシステイン残基中のスルフィド基が、以下の式: In one embodiment, the IgG-binding peptide described herein has the following formula V:
DC- (Xaa2)-(Xaa3)-(Xaa4)-(Xaa1) -G- (Xaa5) -L- (Xaa6) -WCT (V)
(Where
D is an aspartic acid residue;
C is a cysteine residue;
G is a glycine residue;
L is a leucine residue;
W is a tryptophan residue;
T is a threonine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
Xaa2 is an alanine residue, a serine residue or a threonine residue,
Xaa3 is a tryptophan residue or a tyrosine residue,
Xaa4 is a histidine residue, arginine residue, serine residue or threonine residue,
Xaa5 is a glutamic acid residue, an asparagine residue, an arginine residue, or an aspartic acid residue; and
Xaa6 is an isoleucine residue or a valine residue), and the sulfide group in the two cysteine residues outside the peptide is represented by the following formula:
19)DCTYT(Xaa1)GNLVWCT(配列番号19)、
20)DCAYT(Xaa1)GNLVWCT(配列番号20)、
21)DCSYT(Xaa1)GNLVWCT(配列番号21)、
22)DCTWT(Xaa1)GNLVWCT(配列番号22)、
23)DCTYH(Xaa1)GNLVWCT(配列番号23)、
24)DCTYR(Xaa1)GNLVWCT(配列番号24)、
25)DCTYS(Xaa1)GNLVWCT(配列番号25)、
26)DCTYT(Xaa1)GNLVWCT(配列番号26)、
27)DCTYT(Xaa1)GELVWCT(配列番号27)、
28)DCTYT(Xaa1)GRLVWCT(配列番号28)、
29)DCTYT(Xaa1)GDLVWCT(配列番号29)、及び
30)DCTYT(Xaa1)GNLIWCT(配列番号30)
(式中、Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体である)。 Some specific examples of the peptide of formula V are listed below in 19) -30), but it goes without saying:
19) DCTYT (Xaa1) GNLVWCT (SEQ ID NO: 19),
20) DCAYT (Xaa1) GNLVWCT (SEQ ID NO: 20),
21) DCSYT (Xaa1) GNLVWCT (SEQ ID NO: 21),
22) DCTWT (Xaa1) GNLVWCT (SEQ ID NO: 22),
23) DCTYH (Xaa1) GNLVWCT (SEQ ID NO: 23),
24) DCTYR (Xaa1) GNLVWCT (SEQ ID NO: 24),
25) DCTYS (Xaa1) GNLVWCT (SEQ ID NO: 25),
26) DCTYT (Xaa1) GNLVWCT (SEQ ID NO: 26),
27) DCTYT (Xaa1) GELVWCT (SEQ ID NO: 27),
28) DCTYT (Xaa1) GRLVWCT (SEQ ID NO: 28),
29) DCTYT (Xaa1) GDLVWCT (SEQ ID NO: 29) and 30) DCTYT (Xaa1) GNLIWCT (SEQ ID NO: 30)
(Wherein Xaa1 is an arginine residue, a lysine residue, a leucine residue, an asparagine residue, or a derivative thereof).
システイン残基を2つ含むペプチドと、上記リンカーの波線部分に架橋反応に関与する反応性の官能基(例えば、ハロゲン基、イミダゾール基等)を有する化合物を、例えば酸性条件下で混合する工程を含む方法により得ることができる。 The method for preparing the peptide having the linker is not particularly limited. For example, the following formula:
A step of mixing a peptide containing two cysteine residues and a compound having a reactive functional group (for example, a halogen group, an imidazole group, etc.) involved in a crosslinking reaction in the wavy portion of the linker, for example, under acidic conditions It can be obtained by the method of including.
一態様において、本発明は、上記のIgG結合ペプチドを含む固相担体を含む、IgG、好ましくはヒトIgG分離用カラムに関する。 <IgG separation column or kit for purification of IgG>
In one aspect, the present invention relates to a column for separating IgG, preferably human IgG, comprising a solid phase carrier comprising the above-mentioned IgG-binding peptide.
一態様において、本発明は、上記固相担体、又は上記IgG分離用カラムにIgGを結合させる工程、及び結合したIgGを溶出させてIgGを回収する工程を含む、IgG、好ましくはヒトIgGの精製方法に関する。 <Method for purifying IgG>
In one embodiment, the present invention includes the step of binding IgG to the solid phase carrier or the column for separating IgG, and the step of eluting the bound IgG and recovering IgG, preferably purification of human IgG Regarding the method.
N末端をビオチン化PEG4でブロックした以下の6つのIgG結合ペプチドをFmoc固相合成法により常法に従って合成した:
DCAYH(Xaa1)GELVWCT(配列番号1、ただしXaa1はアルギニンで、C末端はアミド化);
GPRCAYH(Xaa1)GELVWCSFH(配列番号4、ただしXaa1はアルギニンで、C末端はアミド化);
GPDCAYH(Xaa1)GELVWCTFH(配列番号2、ただしXaa1はアルギニンで、C末端はアミド化);
GPDCAYH(Xaa1)GELVWCTFH(配列番号2、ただしXaa1はロイシンで、C末端はアミド化);GPDCAYH(Xaa1)GELVWCTFH(配列番号2、ただしXaa1はリシンで、C末端はアミド化);
GPDCAYH(Xaa1)GELVWCTFH(配列番号2、ただしXaa1はアセチル化リシンで、C末端はアミド化)。 [Example 1: Preparation of IgG-binding peptide and measurement of binding affinity]
The following 6 IgG-binding peptides, whose N-termini were blocked with biotinylated PEG4, were synthesized according to a conventional method by Fmoc solid phase synthesis:
DCAYH (Xaa1) GELVWCT (SEQ ID NO: 1, where Xaa1 is arginine and the C-terminal is amidated);
GPRCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 4, where Xaa1 is arginine and the C-terminus is amidated);
GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2, where Xaa1 is arginine and the C-terminal is amidated);
GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2, where Xaa1 is leucine and C-terminal is amidated); GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2, where Xaa1 is lysine and C-terminal is amidated);
GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2, where Xaa1 is acetylated lysine and the C-terminal is amidated).
NH2-PEG4化合成ペプチドGPDCAYH(Xaa1)GELVWCTFH(配列番号2、Xaa1はアルギニンで、C末端はアミド化)は、ペプチド合成ビーズ(Rink-amide-Chemmatrix resin、Biotage)上にて、Fmoc固相合成法により常法に従って合成した。 [Example 2: Preparation of IgG-binding peptide cross-linked by linker]
NH2-PEG4 synthetic peptide GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2, Xaa1 is arginine, C-terminal is amidated) is synthesized on Fmoc solid phase synthesis on peptide synthetic beads (Rink-amide-Chemmatrix resin, Biotage) The compound was synthesized according to a conventional method.
ジクロロアセトン架橋ペプチドが、ヒト抗体精製アフィニティーリガンドとして利用可能か、NHS活性化プレパックカラム (GE Healthcare)に固定化し、各種評価を実施した。 [Example 3: Separation of γ-globulin derived from human serum]
Whether the dichloroacetone cross-linked peptide can be used as a human antibody purified affinity ligand was immobilized on an NHS-activated prepacked column (GE Healthcare) and subjected to various evaluations.
実施例3と同じ方法で作製したカラムに対し、1 mgのヒト由来血清γ-グロブリンを送液し、吸着させた。カラムを吸着溶液で洗浄後、吸着溶液に溶解した0.4 mg/mLの実施例2で調製したペプチドaの溶液を2.5 mL送液した。0.5 mL分画した各フラクションを定法に従い、還元条件下でのSDS-PAGEに供した。タンパク質の検出は、CBB染色で行った。比較のため、クエン酸溶出でも同様の操作を行った。 [Example 4: Competitive elution with peptide]
1 mg of human-derived serum γ-globulin was fed to the column prepared in the same manner as in Example 3 and adsorbed. After washing the column with an adsorption solution, 2.5 mL of 0.4 mg / mL peptide a solution prepared in Example 2 dissolved in the adsorption solution was fed. Each fraction fractioned with 0.5 mL was subjected to SDS-PAGE under reducing conditions according to a conventional method. Protein detection was performed by CBB staining. For comparison, the same operation was performed for citric acid elution.
実施例3と同様の方法で、ペプチド固定化量の異なる3種 (1 mg, 4 mg, 10 mg)のカラムを作製した。ただし、ペプチド溶液は、4 mg/mL、10 mg/mLを準備し、固定化に用いた。 [Example 5: Dynamic binding capacity (DBC) measurement]
In the same manner as in Example 3, three types of columns (1 mg, 4 mg, 10 mg) with different amounts of peptide immobilization were prepared. However, 4 mg / mL and 10 mg / mL peptide solutions were prepared and used for immobilization.
実施例3と同じ方法で作製した1 mgペプチド固定化1 mLカラムに、0.1 M 水酸化ナトリウム溶液を5 mL送液した。その後、吸着溶液で洗浄を行い、実施例5と同様に、流速1 mL/minでのDBC測定を行った。続いて、水酸化ナトリウム溶液処理/DBC測定を5回繰り返し行うことでアルカリ耐性を評価した。水酸化ナトリウム処理を行う前のDBCを100%とし、DBC変動率を求めた。 [Example 6: Evaluation of alkali resistance]
5 mL of a 0.1 M sodium hydroxide solution was fed to a 1 mg peptide-immobilized 1 mL column prepared in the same manner as in Example 3. Thereafter, the adsorbed solution was washed, and DBC measurement was performed at a flow rate of 1 mL / min in the same manner as in Example 5. Subsequently, alkali resistance was evaluated by repeating sodium hydroxide solution treatment /
比較例1として、ジスルフィド結合で架橋されたペプチド を1 mg固定化したカラムを作製し、実施例5と同様にアルカリ耐性評価を実施した。
実施例5及び比較例1の結果を図4に示し、実測値を表3にまとめた。 [Comparative Example 1]
As Comparative Example 1, a column was prepared by immobilizing 1 mg of a peptide crosslinked with a disulfide bond, and alkali resistance was evaluated in the same manner as in Example 5.
The results of Example 5 and Comparative Example 1 are shown in FIG. 4, and the measured values are summarized in Table 3.
Claims (19)
- ヒトIgGと結合可能であることを特徴とするペプチドを固定化した固相担体であって、
前記ペプチドが、下記の式I:
(X1-3)-C-(X2)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(X1-3) (I)
(式中、Xの各々は独立的にシステイン以外の任意のアミノ酸残基であり、
Cはシステイン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Xaa2はグルタミン酸残基又はアスパラギン残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、かつ
Wはトリプトファン残基である。)
によって表される、13~17アミノ酸残基からなるアミノ酸配列を含み、かつ前記ペプチドの外側の2つのシステイン残基中のスルフィド基が、以下の式:
Said peptide is represented by the following formula I:
(X 1-3 ) -C- (X 2 ) -H- (Xaa1) -G- (Xaa2) -LVWC- (X 1-3 ) (I)
(In the formula, each X is independently any amino acid residue other than cysteine,
C is a cysteine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
Xaa2 is a glutamic acid residue or an asparagine residue,
L is a leucine residue;
V is a valine residue, and
W is a tryptophan residue. )
The sulfide group in the two cysteine residues outside the peptide comprising an amino acid sequence consisting of 13-17 amino acid residues represented by
- 前記ペプチドが、下記の式II:
(X1-3)-C-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(X1-3) (II)
(式中、Xの各々は独立的にシステイン以外の任意のアミノ酸残基であり、
Cはシステイン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Xaa2はグルタミン酸残基又はアスパラギン残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、
Wはトリプトファン残基であり、
Xaa3はアラニン残基、セリン残基又はトレオニン残基であり、かつ
Xaa4はチロシン残基又はトリプトファン残基である。)
によって表される、13~17アミノ酸残基からなるアミノ酸配列を含む、請求項1に記載の固相担体。 The peptide is represented by the following formula II:
(X 1-3 ) -C- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -LVWC- (X 1-3 ) (II)
(In the formula, each X is independently any amino acid residue other than cysteine,
C is a cysteine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
Xaa2 is a glutamic acid residue or an asparagine residue,
L is a leucine residue;
V is a valine residue;
W is a tryptophan residue;
Xaa3 is an alanine residue, a serine residue or a threonine residue, and
Xaa4 is a tyrosine residue or a tryptophan residue. )
The solid phase carrier according to claim 1, comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by: - 前記ペプチドが、下記の式III:
(X1-3)-C-A-Y-H-(Xaa1)-G-E-L-V-W-C-(X1-3) (III)
(式中、Xの各々は独立的にシステイン以外の任意のアミノ酸残基であり、
Cはシステイン残基であり、
Aはアラニン残基であり、
Yはチロシン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Eはグルタミン酸残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、かつ
Wはトリプトファン残基である。)
によって表される、13~17アミノ酸残基からなるアミノ酸配列を含む、請求項1又は2に記載の固相担体。 Said peptide is represented by formula III:
(X 1-3 ) -CAYH- (Xaa1) -GELVWC- (X 1-3 ) (III)
(In the formula, each X is independently any amino acid residue other than cysteine,
C is a cysteine residue;
A is an alanine residue,
Y is a tyrosine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
E is a glutamic acid residue,
L is a leucine residue;
V is a valine residue, and
W is a tryptophan residue. )
The solid phase carrier according to claim 1, comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by: - 17アミノ酸残基とした場合の、前記ペプチドのN末端から1~3、15~17番目の各アミノ酸残基が、
1番目のアミノ酸残基= S、G、F又は、なし
2番目のアミノ酸残基= D、G、A、S、P、ホモシステイン、又は、なし
3番目のアミノ酸残基= S、D、T、N、E又はR、
15番目のアミノ酸残基= S、T又はD、
16番目のアミノ酸残基= H、G、Y、T、N、D、F、ホモシステイン、又は、なし、
17番目のアミノ酸残基= Y、F、H、M又は、なし
である、請求項1~3のいずれか一項に記載の固相担体。 When the amino acid residues are 17 amino acid residues, the 1st to 3rd and 15th to 17th amino acid residues from the N-terminus of the peptide are
1st amino acid residue = S, G, F or none
2nd amino acid residue = D, G, A, S, P, homocysteine or none
3rd amino acid residue = S, D, T, N, E or R,
15th amino acid residue = S, T or D,
16th amino acid residue = H, G, Y, T, N, D, F, homocysteine or none,
The solid phase carrier according to any one of claims 1 to 3, wherein the 17th amino acid residue = Y, F, H, M or none. - 前記ペプチドが、以下の1)~14)のいずれかのアミノ酸配列からなる、ただし、Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、Xaa2はホモシステインである、請求項4に記載の固相担体:
1)DCAYH(Xaa1)GELVWCT(配列番号1)
2)GPDCAYH(Xaa1)GELVWCTFH(配列番号2)
3)RCAYH(Xaa1)GELVWCS(配列番号3)
4)GPRCAYH(Xaa1)GELVWCSFH(配列番号4)
5)SPDCAYH(Xaa1)GELVWCTFH(配列番号5)
6)GDDCAYH(Xaa1)GELVWCTFH(配列番号6)
7)GPSCAYH(Xaa1)GELVWCTFH(配列番号7)
8)GPDCAYH(Xaa1)GELVWCSFH(配列番号8)
9)GPDCAYH(Xaa1)GELVWCTHH(配列番号9)
10)GPDCAYH(Xaa1)GELVWCTFY(配列番号10)
11)SPDCAYH(Xaa1)GELVWCTFY(配列番号11)
12)SDDCAYH(Xaa1)GELVWCTFY(配列番号12)
13)RGNCAYH(Xaa1)GQLVWCTYH(配列番号13)
14)G(Xaa2)DCAYH(Xaa1)GELVWCT(Xaa2)H(配列番号14)。 The peptide consists of any one of the following amino acid sequences 1) to 14), wherein Xaa1 is an arginine residue, lysine residue, leucine residue, or asparagine residue, or a derivative thereof, and Xaa2 is The solid phase carrier according to claim 4, which is homocysteine:
1) DCAYH (Xaa1) GELVWCT (SEQ ID NO: 1)
2) GPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 2)
3) RCAYH (Xaa1) GELVWCS (SEQ ID NO: 3)
4) GPRCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 4)
5) SPDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 5)
6) GDDCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 6)
7) GPSCAYH (Xaa1) GELVWCTFH (SEQ ID NO: 7)
8) GPDCAYH (Xaa1) GELVWCSFH (SEQ ID NO: 8)
9) GPDCAYH (Xaa1) GELVWCTHH (SEQ ID NO: 9)
10) GPDCAYH (Xaa1) GELVWCTFY (SEQ ID NO: 10)
11) SPDCAYH (Xaa1) GELVWCTFY (SEQ ID NO: 11)
12) SDDCAYH (Xaa1) GELVWCTFY (SEQ ID NO: 12)
13) RGNCAYH (Xaa1) GQLVWCTYH (SEQ ID NO: 13)
14) G (Xaa2) DCAYH (Xaa1) GELVWCT (Xaa2) H (SEQ ID NO: 14). - 前記ペプチドが、下記の式IV:
D-C-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-T (IV)
(式中、
Dはアスパラギン酸残基であり、
Cはシステイン残基であり、
Hはヒスチジン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Gはグリシン残基であり、
Xaa2はグルタミン酸残基又はアスパラギン残基であり、
Lはロイシン残基であり、
Vはバリン残基であり、
Wはトリプトファン残基であり、
Tはトレオニン残基であり、
Xaa3はアラニン残基又はトレオニン残基であり、かつ、
Xaa4はチロシン残基又はトリプトファン残基である。)によって表される、13アミノ酸残基からなるアミノ酸配列を含む、請求項1又は2に記載の固相担体。 Said peptide is represented by formula IV:
DC- (Xaa3)-(Xaa4) -H- (Xaa1) -G- (Xaa2) -LVWCT (IV)
(Where
D is an aspartic acid residue;
C is a cysteine residue;
H is a histidine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
G is a glycine residue;
Xaa2 is a glutamic acid residue or an asparagine residue,
L is a leucine residue;
V is a valine residue;
W is a tryptophan residue;
T is a threonine residue;
Xaa3 is an alanine residue or a threonine residue, and
Xaa4 is a tyrosine residue or a tryptophan residue. The solid phase carrier according to claim 1, comprising an amino acid sequence consisting of 13 amino acid residues represented by - 前記ペプチドが、以下の1)~4)のいずれかのアミノ酸配列からなる、ただし、Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体である、請求項6に記載の固相担体:
1)DCTYH(Xaa1)GNLVWCT(配列番号15)
2)DCAYH(Xaa1)GNLVWCT(配列番号16)
3)DCTYH(Xaa1)GELVWCT(配列番号17)
4)DCAWH(Xaa1)GELVWCT(配列番号18)。 The peptide consists of an amino acid sequence of any one of the following 1) to 4), wherein Xaa1 is an arginine residue, a lysine residue, a leucine residue, an asparagine residue, or a derivative thereof: Solid phase carrier according to 6:
1) DCTYH (Xaa1) GNLVWCT (SEQ ID NO: 15)
2) DCAYH (Xaa1) GNLVWCT (SEQ ID NO: 16)
3) DCTYH (Xaa1) GELVWCT (SEQ ID NO: 17)
4) DCAWH (Xaa1) GELVWCT (SEQ ID NO: 18). - ヒトIgGと結合可能であることを特徴とするペプチドを固定化した固相担体であって、
前記ペプチドが、下記の式V:
D-C-(Xaa2)-(Xaa3)-(Xaa4)-(Xaa1)-G-(Xaa5)-L-(Xaa6)-W-C-T (V)
(式中、
Dはアスパラギン酸残基であり、
Cはシステイン残基であり、
Gはグリシン残基であり、
Lはロイシン残基であり、
Wはトリプトファン残基であり、
Tはトレオニン残基であり、
Xaa1はアルギニン残基、リシン残基、ロイシン残基、若しくはアスパラギン残基、又はそれらの誘導体であり、
Xaa2はアラニン残基、セリン残基又はトレオニン残基であり、
Xaa3はトリプトファン残基又はチロシン残基であり、
Xaa4はヒスチジン残基、アルギニン残基、セリン残基又はトレオニン残基であり、
Xaa5はグルタミン酸残基、アスパラギン残基、アルギニン残基、又はアスパラギン酸残基であり、かつ
Xaa6はイソロイシン残基又はバリン残基である)によって表される、13アミノ酸残基からなるアミノ酸配列を含み、かつ前記ペプチドの外側の2つのシステイン残基中のスルフィド基が、以下の式:
Said peptide is represented by the following formula V:
DC- (Xaa2)-(Xaa3)-(Xaa4)-(Xaa1) -G- (Xaa5) -L- (Xaa6) -WCT (V)
(Where
D is an aspartic acid residue;
C is a cysteine residue;
G is a glycine residue;
L is a leucine residue;
W is a tryptophan residue;
T is a threonine residue;
Xaa1 is an arginine residue, a lysine residue, a leucine residue, or an asparagine residue, or a derivative thereof,
Xaa2 is an alanine residue, a serine residue or a threonine residue,
Xaa3 is a tryptophan residue or a tyrosine residue,
Xaa4 is a histidine residue, arginine residue, serine residue or threonine residue,
Xaa5 is a glutamic acid residue, an asparagine residue, an arginine residue, or an aspartic acid residue; and
Xaa6 is an isoleucine residue or a valine residue), and the sulfide group in the two cysteine residues outside the peptide is represented by the following formula:
- Xaa1がアルギニン残基、リシン残基若しくはそのアシル化誘導体、又はロイシン残基である、請求項1~8のいずれか一項に記載の固相担体。 The solid phase carrier according to any one of claims 1 to 8, wherein Xaa1 is an arginine residue, a lysine residue or an acylated derivative thereof, or a leucine residue.
- 前記ペプチドが、以下のアミノ酸配列からなる、請求項1に記載の固相担体:
GPDCAYHRGELVWCTFH(配列番号31)。 The solid phase carrier according to claim 1, wherein the peptide consists of the following amino acid sequence:
GPDCAYHRGELVWCTFH (SEQ ID NO: 31). - 前記ペプチドのN末端がPEG化されている、請求項1~11のいずれか一項に記載の固相担体。 The solid phase carrier according to any one of claims 1 to 11, wherein the N-terminus of the peptide is PEGylated.
- 前記ペプチドのC末端がアミド化されている、請求項1~12のいずれか一項に記載の固相担体。 The solid phase carrier according to any one of claims 1 to 12, wherein the C-terminus of the peptide is amidated.
- 前記ペプチドが多量体化されている、請求項1~13のいずれか一項に記載の固相担体。 The solid phase carrier according to any one of claims 1 to 13, wherein the peptide is multimerized.
- 前記ペプチドの多量体が、該ペプチド間にスペーサーを有する、請求項14に記載の固相担体。 The solid phase carrier according to claim 14, wherein the multimer of the peptides has a spacer between the peptides.
- 前記ペプチドと固相の間にスペーサーを有する、請求項1~15のいずれか一項に記載の固相担体。 The solid phase carrier according to any one of claims 1 to 15, which has a spacer between the peptide and the solid phase.
- 請求項1~16のいずれか一項に記載の固相担体を含む、IgG分離用カラム。 An IgG separation column comprising the solid phase carrier according to any one of claims 1 to 16.
- 請求項1~16のいずれか一項に記載の固相担体又は請求項17に記載のIgG分離用カラムを含む、IgGの精製のためのキット。 A kit for purification of IgG comprising the solid phase carrier according to any one of claims 1 to 16 or the IgG separation column according to claim 17.
- 請求項1~16のいずれか一項に記載の固相担体又は請求項17に記載のIgG分離用カラムにIgGを結合させる工程、及び
結合したIgGを溶出させてIgGを回収する工程を含む、IgGの精製方法。
A step of binding IgG to the solid phase carrier according to any one of claims 1 to 16 or the IgG separation column according to claim 17, and a step of recovering IgG by eluting the bound IgG. Purification method of IgG.
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